Existing methods for observing and characterizing living cells such as microorganisms (bacteria, algae, yeasts, etc.) are often invasive and lead to the destruction of the observed sample or to its modification during preparation of the sample—for example, in the case of electron microscopy, water removal occurs under vacuum. This destruction and modification may thus cause artifacts and lead to interpretational problems.
Conventional microscopy techniques based on optical radiation do not allow quantitative information to be obtained on the refractive index or permittivity of samples because they are only capable of detecting the intensity of the light diffracted by these objects, and thus their applicability is restricted in many cases. Their resolution is also diffraction limited.
Digital holographic imaging allows an absolute three-dimensional map to be obtained both of refractive index and permittivity or of the shape of a structured surface (whether transparent or not) with a subwavelength resolution in the visible domain, and therefore with a resolution of less than one micron. Such a tool has many applications in biology and nanotechnology (functionalization of surfaces, polymer films of nanoscale thickness, carbon nanotubes, nanowires, etc.), such as for example 3D tomography of microorganisms, the study of the internal structure of unmarked cells or even quality control in the production of etched components typically of a few hundred nanometers in size. It allows life to be studied in its environment without having to disrupt it (bacterial biofilms in networks for supplying drinking water, pathogenic bacteria in the food processing or health fields, etc.).
An example of a digital holographic microscope based on the principle of what is referred to as “off-axis” holography is described with reference to FIG. 1. It comprises a source 1 of coherent light such as a laser that sends a beam to a first cube beam splitter 2. A first portion of the beam is shaped by means of a collimator 3 including a spatial filter 4, in order to form a reference wave ΣR that is slightly inclined relative to the optical axis (hence the term “off-axis”) and that is detected through a second cube beam splitter 11 by a digital sensor 12 such as a CCD or CMOS camera. The other portion of the beam, issued from the first cube beam splitter, is shaped by means of another collimator 5a-5b including another spatial filter 6, then transmitted (as shown in this figure) or reflected to a sample or object 10. The wave diffracted by this sample is enlarged by means of an objective 9 that, with the second cube beam splitter 11, forms the image of the sample in the proximity of the sensor 12. The diffracted and enlarged wave ΣO interferes with the reference wave ΣR, thus forming a hologram on the sensor 12. This microscope records a digital hologram of the wave diffracted by the object.
Apart from the fact that such a recording configuration is bulky, it is in addition sensitive to external perturbations that modify the optical paths of the reference wave and the wave diffracted by the object and thus degrade the hologram, thereby hindering observation of the object and its reconstruction. This is accentuated during the observation of dynamic effects.
Provided that a hologram is successfully obtained, the amplitude and phase of the object are numerically reconstructed using known methods such as for example described in the publication CUCHE E., MARQUET P., DEPEURSINGE C., “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms”, Applied Optics, Vol. 38, p. 6994-7001, 1999:                by calculating the spectrum of the hologram, for example by applying a discrete Fourier transform (DFT);        by digitally filtering the lobe of the spectrum representative of the amplitude and phase of the object; and        by calculating the inverse DFT of the filtered lobe in order thus to obtain an amplitude image and a phase image of the object.        
A step of numerical focusing is advantageously applied to the images obtained.
The calculation of the complex amplitude of the wave front of the object (also designated the complex amplitude of the wave of the object) in the plane of the sensor, denoted A=a·exp(iφ), may be expressed by the two equations (1) and (2):a=|DFT−1{f[DFT{H}]}|  (1)φ=arg(DFT−1{f[DFT{H}]})  (2)where H is the recorded hologram, a and φ are the modulus and the phase of the complex amplitude, respectively, and f[ . . . ] represents the process of digitally filtering the lobe of the spatial frequency spectrum representative of the amplitude and phase of the object. DFT{ . . . } and DFT−1{ . . . } are the direct and inverse discrete Fourier transform operators, respectively, and arg( . . . ) is the argument of the complex amplitude (i.e. its phase). These equations do not contain correcting terms that could optionally be applied to the calculation.
There is another digital holography technique (less common than off-axis holography) called “in-line” holography, which uses a reference beam oriented in the same direction as the object beam. With this technique, only the amplitude of the object is accessible. This is because the superposition of the object beam ΣO and reference beam ΣR limits the measurement of phase from the recorded holograms. The advantage of this technique in certain applications (study of opaque objects for example) is the simplicity of its implementation as it does not require fine adjustment of the reference beam. It is also possible to bypass the superposition of diffraction orders due to the “in-line” character by modulating the reference wave (what is referred to as the “phase shift” method). However, this approach requires at least three phase-shifted holograms (4 in general) to be recorded in sequence, which limits the method in the study of dynamic effects, as described in the publication YAMAGUCHI I., KATO J., OHTA S., MIZUNO J., “Image formation in phase shifting digital holography and application to microscopy”, Applied Optics, Vol. 40, p. 6177-6186, 2001.
At the present time, there remains a need for a system that is satisfactory both in terms of bulk and sensitivity to exterior perturbations and that is applicable to the study of dynamic effects.